From the art several methods are known that preserve directional information using distinguishable adapters for different ends of double-stranded DNA.
Adapters may be ligated directly to single-stranded RNA molecules.
Special composite primers may be used for first-strand cDNA synthesis: Herein the 5′-part of the primer provides a directional information, the 3′-part (oligo(dT)) or random nucleotides) is able to form a duplex with the RNA. Recently, first-strand cDNA synthesis was performed from a tagged random hexamer primer and second strand synthesis from a DNA-RNA template-switching-primer.
Special homopolymeric adapters may be synthesised at the 3′-end of DNA molecules by Terminal Nucleotide Transferase.
DNA-RNA template-switching is used to attach a specific adapter to 3′-end of the DNA copy of the original single-stranded RNA molecule.
The main disadvantage of the adaptor-based approach is that only the ends of the clones preserve directional information. The problems are:                (i) strand-specific operations (directional immobilization on a solid phase; production of a single-stranded DNA for hybridization, etc.) should act on the adaptor sequence. For long clones the efficiency of such operations is normally low. For example, strand separation where one of the strands contains a biotin molecule at the 5′-end is getting complicated if the length of the duplex is higher than 1 kb.        (ii) after fragmentation internal parts of the clones lose the directional information.        
A special approach which is able to keep directional information in any part of the sequence was suggested recently: All cytidine residues in the RNA were converted to undines by bisulfite treatment prior to cDNA synthesis. The problem is that this approach is laborious and also leads to the loss of about 30% of uniquely matched sequencing reads because a part of the genome complexity is lost during the transformation from the four-bases into the three-bases code.
A lot of biologically important nucleic acids function in a single-stranded form where the polarity is extremely important. But for basic molecular cloning procedures single-stranded polynucleotides should be converted into double-stranded DNA molecules, because                most of restriction enzymes work only on double-stranded DNA;        ligation of double-stranded molecules is much simpler in comparison with the ligation of single-stranded molecules;        the result of PCR is a double-stranded DNA.        
At the moment most of the protocols keep directional information in the form of distinguishable adaptors on the ends of double-stranded molecules (FIG. 1B).
US 2007/0281863 discloses a method for characterizing the amounts of nucleic acids, including plus/minus determinations. To this end the mRNA is transcribed into double stranded cDNA and two different RNA promoters are integrated in both cDNA strands respectively. By using the different RNA promoters in the presence of allylamine-UTP nucleotides plus or minus RNA with modified allylamine-UTP nucleotides is generated. The modified allylamine-UTP nucleotides can be labeled with NHS-Cy3/5 and are then hybridized in different nucleic acid arrays.
The main disadvantage of this approach is that the RNA cannot be used for high-throughput complementary DNA sequencing (RNA-Seq). Direct cDNA sequencing (RNA-Seq) is a new tool for whole-transcriptome analysis. Second generation sequencing machines have increased sequencing throughput by about two orders of magnitude, making global transcriptome sequencing feasible. Since sequencing costs are constantly decreasing (contrary to those of microarrays) cDNA sequencing will capture a considerable portion of transcriptome analyses in the future.
The RNA-Seq procedure is simple, has a large dynamic range and high sensitivity, and can unequivocally identify splicing and RNA editing products as well as allele-specific transcripts. RNA-Seq provides a number of further advantages over other high throughput approaches like microarray hybridization, gene-specific and tiling arrays or SAGE-analyses. The depth of RNA-Seq analyses is flexible, providing a dynamic range typically an order of magnitude greater than one can achieve with hybridization arrays. The digital character of the RNA-Seq data permits to compare and pool results from different laboratories. No prior information about transcripts sequences is required, allowing detection of novel transcripts. It is possible to estimate the absolute level of gene expression and to study structure of transcripts.
However, the weakness of RNA-Seq was the inability to determine the polarity of RNA transcripts without laborious modification of the protocol. The method according to the invention solves this problem allowing the strand specific differentiation with an easy, robust and cheap procedure. The method is less susceptible and more robust compared to the method described in US 2007/0281863 because there is no need to transcribe the mRNA first in cDNA then inserting two different promoters in the cDNA and then using two different polymerases to transcribe the cDNA again in RNA including an additional step to label the marked RNA with NHS-Cy3/5.
Thus, the modified allylamine-UTP RNA according to US 2007/0281863 can only be used in hybridization arrays that are very expensive, less sensitive and cannot detect novel transcripts. Additionally, the method is laborious including the integration of promoters and different polymerases and works with RNA as an end product, which is very susceptible to degradation because of abundant RNases and can only be used in expensive hybridization arrays that are less sensitive then e.g. RNA-Seq.
Further disadvantages include that many basic molecular cloning procedures are performed on double stranded cDNA, which are necessary for many subsequent analyzing methods. The method according to US 2007/0281863 generates a double stranded cDNA with two promoters as an intermediate product. However, these intermediate cDNAs are not preferred for cloning procedures,
As we already explained above the main disadvantage of adaptor-based approaches is that only the ends of the clones preserve directional information. The integration of promoters in the cDNA is basically the same procedure. During cloning procedures the promoters can be changed, damaged etc., especially because most cloning procedures modify the ends of the cDNA. The subsequent strand specific transcription of the cDNA with promoter specific polymerases is thus not very robust and the strand specific information can be lost during molecular cloning procedures. Taking into account the susceptibility of RNA to degradation, the method according to the instant application is more functional, i.e. the RNA is transcribed directly into cDNA and the strand specific information is already integrated in this step. Furthermore, the inventive method is much more robust, e.g. the cDNA already contains the strand specific information along the whole length of the transcript and no molecular cloning procedures can compromise this information. Consequently, the inventive method is convenient, reliable and highly reproducible.
WO 97/12061 discloses a method for characterizing nucleic acid molecules, comprising synthesizing DNA with a nucleic acid matrix, primers, polymerase, four canonical deoxynucleotides and at least one non-canonical deoxynucleotide. The DNA is then contacted with N-glycosylase and the abasic sites are treated in a way to leading to breakage of the phosphodiester backbone. The resulting DNA fragments are separated according to their size.
This method characterizes DNA according to their size by using non-canonical deoxynucleotide to label one synthesized DNA strand, but the method is not able to preserve the information about the direction of the original single-stranded molecules after being transformed into a double-stranded form.